Upon insulin addition, GLUT4 transport vesicles accumulate and fuse at plasma membrane hot spots, creating clusters. To study the state of the GLUT4 molecules in these PM clusters in primary human adipose cells, we introduced a photo-switchable GLUT4 construct, HA-GLUT4-EOS, and applied a novel photo-activation localization microscopy technique, together with total-internal reflection fluorescence microscopy to track single GLUT4 molecules. We detected two distinct classes of GLUT4 molecule motions: unconstrained lateral diffusion and cluster-confined immobilization. We found that single GLUT4 molecules could get trapped in clusters, severely limiting their diffusion. Conversely, GLUT4 molecules were detected leaving their trapped state in these PM clusters (released) and resuming diffusion at 0.1 m2/s. Double-labeling of insulin-responsive vesicles with GLUT4-mCherry and IRAP-pHluorin was used to detect individual fusion events with PM. GLUT4 clusters were formed through fusion of GLUT4-containing vesicles with PM in an insulin-independent way. GLUT4 molecules were retained within the clusters by an unknown mechanism specific to GLUT4, but not to IRAP. Insulin, on the other hand, enhanced the rate of fusion events that released all GLUT4 into PM. These data provide the first evidence of a dynamic exchange of GLUT4 molecules between clusters and PM, and link insulin-dependent and insulin-independent GLUT4 recycling pathways. Moreover, these findings suggest that the amount of GLUT4 present in PM is not merely defined by the rates of exocytosis and endocytosis, but rather relies on GLUT4-specific molecular interactions at clusters that regulate its recycling. Thus we confirm a non-uniform GLUT4 distribution in the plasma membrane of primary human adipose cells and show the dynamic nature of GLUT4 organization into clusters. Previous reports demonstrate that adipose cells from insulin-resistant human subjects exhibit decreased levels of GLUT4 and impaired insulin signaling, but the dynamics of GLUT4 trafficking and the insulin-stimulated translocation of GLUT4 have not been examined. Here we transfect isolated adipose cells from insulin-sensitive and resistant human subjects, and measure GLUT4 vesicle (GSV) trafficking and fusion, and the cell-surface associated distribution of GLUT4 in the basal and insulin-stimulated states. In the absence of insulin, GSV trafficking on u-tubules and the basal level of GVS fusion with the plasma membrane (PM) are comparable to that seen in rat adipose cells, and do not vary with the insulin sensitivity of the donor subject. In response to insulin, GSV trafficking is markedly decreased and GSV fusion to PM is markedly increased in the cells from the insulin-sensitive subjects, again in a manner similar to that in rat adipose cells. However, the insulin responses for both of these parameters diminish progressively with increasing insulin resistance in the donor subjects. Total cell-surface GLUT4, PM- dispersed GLUT4, and the intensity of GLUT4 associated with PM clusters in the basal state, measured by an HA binding assay, gradually decrease with decreasing insulin sensitivity. In addition, the stimulation of these parameters by insulin also decreases with decreasing insulin sensitivity. Further, GLUT4 cluster density in PM, that is the number of clusters per unit surface area, also decreases with decreasing insulin sensitivity, but it is the cells from the insulin-resistant subjects that increase their GLUT4 cluster density in response to insulin. The current use of TIRF microscopy allows us to analyze insulin responsive glucose transport on a cell by cell basis; our observations demonstarte that cells are either individually basal or insulin-stimulated, and that the magnitude of the insulin response is determined by the shift in the number of cells between these two states. Thus, while the acute translocation of GLUT4 to PM through tethering and fusion with release decreases progressively with systemic insulin resistance, the still available mobile GSV are redirected to PM fusion with retention and the formation of additional GLUT4 clusters in the insulin-resistant state. These data suggest a mechanism through which basal glucose transport regulation is maintained even under conditions of marked systemic insulin resistance.